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author:("Fu, xiaoming")
1.  Platelet Rich Plasma Clot Releasate Preconditioning Induced PI3K/AKT/NFκB Signaling Enhances Survival and Regenerative Function of Rat Bone Marrow Mesenchymal Stem Cells in Hostile Microenvironments 
Stem Cells and Development  2013;22(24):3236-3251.
Mesenchymal stem cells (MSCs) have been optimal targets in the development of cell based therapies, but their limited availability and high death rate after transplantation remains a concern in clinical applications. This study describes novel effects of platelet rich clot releasate (PRCR) on rat bone marrow-derived MSCs (BM-MSCs), with the former driving a gene program, which can reduce apoptosis and promote the regenerative function of the latter in hostile microenvironments through enhancement of paracrine/autocrine factors. By using reverse transcription–polymerase chain reaction, immunofluorescence and western blot analyses, we showed that PRCR preconditioning could alleviate the apoptosis of BM-MSCs under stress conditions induced by hydrogen peroxide (H2O2) and serum deprivation by enhancing expression of vascular endothelial growth factor and platelet-derived growth factor (PDGF) via stimulation of the platelet-derived growth factor receptor (PDGFR)/PI3K/AKT/NF-κB signaling pathways. Furthermore, the effects of PRCR preconditioned GFP-BM-MSCs subcutaneously transplanted into rats 6 h after wound surgery were examined by histological and other tests from days 0–22 after transplantation. Engraftment of the PRCR preconditioned BM-MSCs not only significantly attenuated apoptosis and wound size but also improved epithelization and blood vessel regeneration of skin via regulation of the wound microenvironment. Thus, preconditioning with PRCR, which reprograms BM-MSCs to tolerate hostile microenvironments and enhance regenerative function by increasing levels of paracrine factors through PDGFR-α/PI3K/AKT/NF-κB signaling pathways would be a safe method for boosting the effectiveness of transplantation therapy in the clinic.
PMCID: PMC3868358  PMID: 23885779
2.  Treatment of MSCs with Wnt1a-conditioned medium activates DP cells and promotes hair follicle regrowth 
Scientific Reports  2014;4:5432.
Hair loss (alopecia) is a common problem for people. The dermal papilla is the key signaling center that regulates hair growth and it engage in crosstalk with the microenvironment, including Wnt signaling and stem cells. In this study, we explored the effects of bone marrow mesenchymal stem cell overexpression of Wnt1a on mouse hair follicle regeneration. Wnt-CM accelerated hair follicle progression from telogen to anagen and enhanced the ALP expression in the DP area. Moreover, the hair induction-related genes were upregulated, as demonstrated by qRT-PCR. Wnt-CM treatment restored and increased DP cell expression of genes downregulated by dihydrotestosterone treatment, as demonstrated by qRT-PCR assays. Our study reveals that BM-MSC-generated Wnt1a promotes the DP's ability to induce hair cycling and regeneration.
PMCID: PMC4069670  PMID: 24961246
3.  Low-Dose Decitabine-Based Chemoimmunotherapy for Patients with Refractory Advanced Solid Tumors: A Phase I/II Report 
Journal of Immunology Research  2014;2014:371087.
Aberrant DNA methylation is one of the main drivers of tumor initiation and progression. The reversibility of methylation modulation makes it an attractive target for novel anticancer therapies. Clinical studies have demonstrated that high-dose decitabine, a hypomethylating agent, results in some clinical benefits in patients with refractory advanced tumors; however, they are extremely toxic. Low doses of decitabine minimize toxicity while potentially improving the targeted effects of DNA hypomethylation. Based on these mechanisms, low-dose decitabine combined with chemoimmunotherapy may be a new treatment option for patients with refractory advanced tumors. We proposed the regimen of low-dose decitabine-based chemoimmunotherapy for patients with refractory advanced solid tumors. A favorable adverse event profile was observed in our trial that was highlighted by the finding that most of these adverse events were grades 1-2. Besides, the activity of our cohort was optimistic and the clinical benefit rate was up to 60%, and the median PFS was prolonged compared with PFS to previous treatment. We also identified a significant correlation between the PFS to previous treatment and clinical response. The low-dose DAC decitabine-based chemoimmunotherapy might be a promising protocol for improving the specificity and efficiency of patients with refractory advanced solid tumors. This trial is registered in the database (identifier NCT01799083).
PMCID: PMC4054619  PMID: 24963497
4.  Nerve growth factor improves functional recovery by inhibiting endoplasmic reticulum stress-induced neuronal apoptosis in rats with spinal cord injury 
Endoplasmic reticulum (ER) stress-induced apoptosis plays a major role in various diseases, including spinal cord injury (SCI). Nerve growth factor (NGF) show neuroprotective effect and improve the recovery of SCI, but the relations of ER stress-induced apoptosis and the NGF therapeutic effect in SCI still unclear.
Young adult female Sprague-Dawley rats’s vertebral column was exposed and a laminectomy was done at T9 vertebrae and moderate contusion injuries were performed using a vascular clip. NGF stock solution was diluted with 0.9% NaCl and administered intravenously at a dose of 20 μg/kg/day after SCI and then once per day until they were executed. Subsequently, the rats were executed at 1d, 3 d, 7d and 14d. The locomotor activities of SCI model rats were tested by the 21-point Basso-Beattie-Bresnahan (BBB) locomotion scale, inclined plane test and footprint analysis. In addition, Western blot analysis was performed to identify the expression of ER-stress related proteins including CHOP, GRP78 and caspase-12 both in vivo and in vitro. The level of cell apoptosis was determined by TUNEL in vivo and Flow cytometry in vitro. Relative downstream signals Akt/GSK-3β and ERK1/2were also analyzed with or without inhibitors in vitro.
Our results demonstrated that ER stress-induced apoptosis was involved in the injury of SCI model rats. NGF administration improved the motor function recovery and increased the neurons survival in the spinal cord lesions of the model rats. NGF decreases neuron apoptosis which measured by TUNEL and inhibits the activation of caspase-3 cascade. The ER stress-induced apoptosis response proteins CHOP, GRP78 and caspase-12 are inhibited by NGF treatment. Meanwhile, NGF administration also increased expression of growth-associated protein 43 (GAP43). The administration of NGF activated downstream signals Akt/GSK-3β and ERK1/2 in ER stress cell model in vitro.
The neuroprotective role of NGF in the recovery of SCI is related to the inhibition of ER stress-induced cell death via the activation of downstream signals, also suggested a new trend of NGF translational drug development in the central neural system injuries which involved in the regulation of chronic ER stress.
PMCID: PMC4039547  PMID: 24884850
Nerve growth factor; Endoplasmic reticulum stress; Spinal cord injury; Apoptosis; Akt/GSK-3β; ERK1/2
5.  Autologous CIK Cell Immunotherapy in Patients with Renal Cell Carcinoma after Radical Nephrectomy 
Objective. To evaluate the efficacy of autologous cytokine-induced killer (CIK) cells in patients with renal cell carcinoma (RCC). Methods. 20 patients diagnosed with TNM stage I or II RCC were randomly divided into two groups, a CIK cell treatment group and a control group. The endpoint was progression-free survival (PFS) evaluated by Kaplan-Meier analyses. Results. CD3+, CD3+/CD8+, CD3+/CD4+, and CD3+/CD56+ levels increased after CIK cell culture (P < 0.01). The median PFS in CIK cell treatment group was significantly longer than that in control group (PFS, 32.2 months versus 21.6 months; log-rank, P = 0.032), all patients were alive during the course of followup, and there are no statistically significant differences between two groups in OS (log-rank, P = 0.214). Grade III or greater adverse events were not observed. Conclusions. CIK cells treatment could prolong survival in patients with RCC after radical nephrectomy and showed acceptable curative effect with potential enhancement of cellular immune function. This trial is registered with NCT01799083.
PMCID: PMC3872096  PMID: 24382970
6.  Infusion of Mesenchymal Stem Cells Ameliorates Hyperglycemia in Type 2 Diabetic Rats 
Diabetes  2012;61(6):1616-1625.
Infusion of mesenchymal stem cells (MSCs) has been shown to effectively lower blood glucose in diabetic individuals, but the mechanism involved could not be adequately explained by their potential role in promoting islet regeneration. We therefore hypothesized that infused MSCs might also contribute to amelioration of the insulin resistance of peripheral insulin target tissues. To test the hypothesis, we induced a diabetic rat model by high-fat diet/streptozotocin (STZ) administration, performed MSC infusion during the early phase (7 days) or late phase (21 days) after STZ injection, and then evaluated the therapeutic effects of MSC infusion and explored the possible mechanisms involved. MSC infusion ameliorated hyperglycemia in rats with type 2 diabetes (T2D). Infusion of MSCs during the early phase not only promoted β-cell function but also ameliorated insulin resistance, whereas infusion in the late phase merely ameliorated insulin resistance. Infusion of MSCs resulted in an increase of GLUT4 expression and an elevation of phosphorylated insulin receptor substrate 1 (IRS-1) and Akt (protein kinase B) in insulin target tissues. This is the first report of MSC treatment improving insulin sensitivity in T2D. These data indicate that multiple roles and mechanisms are involved in the efficacy of MSCs in ameliorating hyperglycemia in T2D.
PMCID: PMC3357293  PMID: 22618776
7.  Culturing on Wharton's Jelly Extract Delays Mesenchymal Stem Cell Senescence through p53 and p16INK4a/pRb Pathways 
PLoS ONE  2013;8(3):e58314.
Mesenchymal stem cells (MSCs) hold great therapeutic potential. However, MSCs undergo replication senescence during the in vitro expansion process. Wharton's jelly from the human umbilical cord harbors a large number of MSCs. In this study, we hypothesized that Wharton's jelly would be beneficial for in vitro expansion of MSCs. Wharton's jelly extract (WJEs), which is mainly composed of extracellular matrix and cytokines, was prepared as coating substrate. Human MSCs were isolated and cultured on WJE-coated plates. Although the proliferation capacity of cells was not augmented by WJE in early phase culture, adynamic growth in late-phase culture was clearly reduced, suggesting that the replicative senescence of MSCs was efficiently slowed by WJE. This was confirmed by β-galactosidase staining and telomere length measurements of MSCs in late-phase culture. In addition, the decreased differentiation ability of MSCs after long-term culture was largely ameliorated by WJE. Reactive oxygen species (ROS), p53, and p16INK4a/pRb expression increased with passaging. Analysis at the molecular level revealed that WJE-based culture efficiently suppressed the enhancement of intracellular ROS, p53, and p16INK4a/pRb in MSCs. These data demonstrated that WJE provided an ideal microenvironment for MSCs culture expansion in vitro preserved MSC properties by delaying MSCs senescence, and allowed large numbers of MSCs to be obtained for basic research and clinical therapies.
PMCID: PMC3596399  PMID: 23516461
8.  Predictors of retention in community-based methadone maintenance treatment program in Pearl River Delta, China 
The aims were to identify predictors of treatment retention in methadone maintenance treatment (MMT) clinics in Pearl River Delta, China.
Retrospective longitudinal study. Participants: 6 MMT clinics in rural and urban area were selected. Statistical analysis: Stratified random sampling was employed, and the data were analyzed using Kaplan-Meier survival curves and life table method. Protective or risk factors were explored using Cox’s proportional hazards model. Independent variables were enrolled in univariate analysis and among which significant variables were analyzed by multivariate analysis.
A total of 2728 patients were enrolled. The median of the retention duration was 13.63 months, and the cumulative retention rates at 1,2,3 years were 53.0%, 35.0%, 20.0%, respectively. Multivariate Cox analysis showed: age, relationship with family, live on support from family or friends, income, considering treatment cost suitable, considering treatment open time suitable, addiction severity (daily expense for drug), communication with former drug taking peer, living in rural area, daily treatment dosage, sharing needles, re-admission and history of being arrested were predictors for MMT retention.
MMT retention rate in Guangdong was low and treatment skills and quality should be improved. Meanwhile, participation of family and society should be encouraged.
PMCID: PMC3599968  PMID: 23497263
Methadone maintenance treatment; Retention; Cox’s proportional hazards model
9.  Characterization of Adult α- and β-Globin Elevated by Hydrogen Peroxide in Cervical Cancer Cells That Play A Cytoprotective Role Against Oxidative Insults 
PLoS ONE  2013;8(1):e54342.
Hemoglobin (Hgb) is the main oxygen and carbon dioxide carrier in cells of erythroid lineage and is responsible for oxygen delivery to the respiring tissues of the body. However, Hgb is also expressed in nonerythroid cells. In the present study, the expression of Hgb in human uterine cervix carcinoma cells and its role in cervical cancer were investigated.
The expression level of Hgb in cervical cancer tissues was assessed by quantitative reverse transcriptase-PCR (qRT-PCR). We applied multiple methods, such as RT-PCR, immunoblotting, and immunohistochemical analysis, to confirm Hgb expression in cervical cancer cells. The effects of ectopic expression of Hgb and Hgb mutants on oxidative stress and cell viability were investigated by cellular reactive oxygen species (ROS) analysis and lactate dehydrogenase (LDH) array, respectively. Both Annexin V staining assay by flow cytometry and caspase-3 activity assay were used, respectively, to evaluate cell apoptosis.
qRT-PCR analysis showed that Hgb-α- (HBA1) and Hgb-β-globin (HBB) gene expression was significantly higher in cervical carcinoma than in normal cervical tissues, whereas the expression of hematopoietic transcription factors and erythrocyte specific marker genes was not increased. Immunostaining experiments confirmed the expression of Hgb in cancer cells of the uterine cervix. Hgb mRNA and protein were also detected in the human cervical carcinoma cell lines SiHa and CaSki, and Hgb expression was up-regulated by hydrogen peroxide-induced oxidative stress. Importantly, ectopic expression of wild type HBA1/HBB or HBA1, rather than mutants HBA1H88R/HBBH93R unable to bind hemo, suppressed oxidative stress and improved cell viability.
The present findings show for the first time that Hgb is expressed in cervical carcinoma cells and may act as an antioxidant, attenuating oxidative stress-induced damage in cervical cancer cells. These data provide a significant impact not only in globin biology but also in understanding of cervical cancer pathogenesis associated with oxidative stress.
PMCID: PMC3547883  PMID: 23349856
10.  A MicroRNA Component of the Neoplastic Microenvironment: Microregulators with Far-Reaching Impact 
BioMed Research International  2012;2013:762183.
The interplay between tumor cells and their microenvironment plays a pivotal role in tumor development and progression. Although a growing body of evidence has established the importance of the tumor microenvironment, an understanding of the crosstalk between its components and cancer cells remains elusive. The pathways triggered by microenvironmental factors could modulate cancer-related gene transcription, also affecting small noncoding RNAs, microRNAs, which have emerged as key posttranscriptional regulators of gene expression, directly involved in human cancers. Although microRNAs regulate most biological mechanisms, their role in the tumor microenvironment has only recently become the focus of intense research. In this paper, we focus on the intertwined connection between the tumor microenvironment and aberrant expression of microRNAs involved in carcinogenesis. We also discuss the emerging roles of microRNAs in the tumor microenvironment as it relates to cancer progression. We conclude that microRNAs are critical for our understanding of the development of cancer, and that targeting microRNA signaling pathways in the microenvironment as well as in tumor cells opens new therapeutic avenues to the global control of cancer.
PMCID: PMC3591172  PMID: 23509776
11.  A Molecular Link Between Interleukin 22 and Intestinal Mucosal Wound Healing 
Advances in Wound Care  2012;1(6):231-237.
Interleukin 22 (IL-22) and signal transducer and activator of transcription 3 (STAT3) are two important regulators of inflammation. Crohn's disease and ulcerative colitis are considered inflammatory bowel diseases (IBDs), due to the belief that these diseases result from dysregulated responses of the intestinal immune system to bacteria present in the commensal flora.
The Problem
It is debated whether a breakdown of immune tolerance is the primary cause of these diseases or occurs downstream of an initial defect of the intestinal barrier and intestinal epithelial cells (IECs).
Basic/Clinical Science Advances
Recent reports suggest a crucial role for IL-22 in the regulation of gut inflammation as well as epithelial barrier integrity. Local IL-22 gene delivery enhances expression of its downstream effector, STAT3, within colonic epithelial cells and induces both STAT3-dependent expression of mucus-associated molecules and restitution of mucus-producing goblet cells. IEC-specific deletion of STAT3 results in significant susceptibility to experimental colitis with a striking defect in epithelial restitution. STAT3 activation, thus, may regulate immune homeostasis in the gut by promoting IL-22-dependent mucosal wound healing.
Clinical Care Relevance
The importance of IL-22/STAT3 signaling in IEC wound healing suggests a critical role for epithelial homeostasis in IBDs.
Effective healing of the IECs could be considered a primary target in the development of treatments for IBDs. IL-22/STAT3 signaling exerts a protective role in the process of intestinal mucosal wound healing and may thereby provide a promising therapeutic approach to the treatment of IBDs.
PMCID: PMC3623586  PMID: 24527311
12.  A Facile and Specific Assay for Quantifying MicroRNA by an Optimized RT-qPCR Approach 
PLoS ONE  2012;7(10):e46890.
The spatiotemporal expression patterns of microRNAs (miRNAs) are important to the verification of their predicted function. RT-qPCR is the accepted technique for the quantification of miRNA expression; however, stem-loop RT-PCR and poly(T)-adapter assay, the two most frequently used methods, are not very convenient in practice and have poor specificity, respectively.
We have developed an optimal approach that integrates these two methods and allows specific and rapid detection of tiny amounts of sample RNA and reduces costs relative to other techniques. miRNAs of the same sample are polyuridylated and reverse transcribed into cDNAs using a universal poly(A)-stem-loop RT primer and then used as templates for SYBR® Green real-time PCR. The technique has a dynamic range of eight orders of magnitude with a sensitivity of up to 0.2 fM miRNA or as little as 10 pg of total RNA. Virtually no cross-reaction is observed among the closely-related miRNA family members and with miRNAs that have only a single nucleotide difference in this highly specific assay. The spatial constraint of the stem-loop structure of the modified RT primer allowed detection of miRNAs directly from cell lysates without laborious total RNA isolation, and the poly(U) tail made it possible to use multiplex RT reactions of mRNA and miRNAs in the same run.
The cost-effective RT-qPCR of miRNAs with poly(A)-stem-loop RT primer is simple to perform and highly specific, which is especially important for samples that are precious and/or difficult to obtain.
PMCID: PMC3465266  PMID: 23071657
13.  Difference in Risk Behaviors and STD Prevalence Between Street-Based and Establishment-Based FSWs in Guangdong Province, China 
AIDS and Behavior  2012;16(4):943-951.
The major mode of HIV/AIDS transmission in China is now heterosexual activities, but risk for HIV and sexually transmitted diseases (STDs) may differ among different strata of female sex workers (FSWs). Respondent-driven sampling was used to recruit 320 FSWs in Guangdong Province, China. The respondents were interviewed using a structured questionnaire, and tested for HIV, syphilis, gonorrhea, and Chlamydia. The street-based FSWs had lower education levels, a higher proportion supporting their families, charged less for their services, and had engaged in commercial sex for a longer period of time than establishment-based FSWs. The proportion consistently using condoms with clients and with regular non-paying partners was also lower. The prevalence of syphilis, gonorrhea, and Chlamydia was higher among street-based sex workers. Being a street-based sex worker, having regular non-paying sex partners, and having non-regular non-paying partners were independent risk factors for inconsistent condom. Street-based FSWs had more risk behaviors than establishment-based FSWs, and should therefore be specifically targeted for HIV as well as STD intervention programs.
PMCID: PMC3338878  PMID: 22228068
Behavior; HIV/AIDS; HIV; STD; Female sex workers
14.  Tissue-engineered skin: bottleneck or breakthrough 
During the past few years, tissue-engineered skin constructs has offered great promise in the treatment of deep burns and various skin-related disorders. The overall impact of bioengineered skin research, as a multidisciplinary field, has also enhanced our understanding of the structure-function relations and the physiological processes of tissue regeneration within these constructed three-dimensional skin models. Despite of the fact that currently available bioengineered skin products have a range of problems such as patient safety, clinical efficacy and convenience of use, they are still one of the most advanced strategies because of their wide use and potential development in the biomedical field. Here, the challenges and developments in tissue-engineered skin research are discussed. Central to the discussion is the extensive application and future prospects of these bioengineered skin constructs.
PMCID: PMC3415937  PMID: 22928152
tissue-engineered skin; biomedicine; biomaterial; cell; wound healing
15.  LRP16 Integrates into NF-κB Transcriptional Complex and Is Required for Its Functional Activation 
PLoS ONE  2011;6(3):e18157.
Nuclear factor κB (NF-κB)-mediated pathways have been widely implicated in cell survival, development and tumor progression. Although the molecular events of determining NF-κB translocation from cytoplasm to nucleus have been extensively documented, the regulatory mechanisms of NF-κB activity inside the nucleus are still poorly understood. Being a special member of macro domain proteins, LRP16 was previously identified as a coactivator of both estrogen receptor and androgen receptor, and as an interactor of NF-κB coactivator UXT. Here, we investigated the regulatory role of LRP16 on NF-κB activation.
GST pull-down and coimmunoprecipitation (CoIP) assays assessed protein-protein interactions. The functional activity of NF-κB was assessed by luciferase assays, changes in expression of its target genes, and its DNA binding ability. Annexin V staining and flow cytometry analysis were used to evaluate cell apoptosis. Immunohistochemical staining of LRP16 and enzyme-linked immunosorbent assay-based evaluation of active NF-κB were performed on primary human gastric carcinoma samples.
We demonstrate that LRP16 integrates into NF-κB transcriptional complex through associating with its p65 component. RNA interference knockdown of the endogenous LRP16 in cells leads to impaired NF-κB activity and significantly attenuated NF-κB-dependent gene expression. Mechanistic analysis revealed that knockdown of LRP16 did not affect tumor necrosis factor α (TNF-α)-induced nuclear translocation of NF-κB, but blunted the formation or stabilization of functional NF-κB/p300/CREB-binding protein transcription complex in the nucleus. In addition, knockdown of LRP16 also sensitizes cells to apoptosis induced by TNF-α. Finally, a positive link between LRP16 expression intensity in nuclei of tumor cells and NF-κB activity was preliminarily established in human gastric carcinoma specimens.
Our findings not only indicate that LRP16 is a crucial regulator for NF-κB activation inside the nucleus, but also suggest that LRP16 may be an important contributor to the aberrant activation of NF-κB in tumors.
PMCID: PMC3069058  PMID: 21483817
16.  Prevalence of Human Immunodeficiency Virus and Sexually Transmitted Infections and Associated Risk Factors among Female Sex Workers in Guangdong Province, China 
To assess the prevalence of human immunodeficiency virus (HIV) and sexually transmitted infections (STIs) and associated factors among female sex workers (FSWs) in Guangdong, China.
Respondent driven sampling was used to recruit 320 FSWs. The recruited participants were interviewed face-to-face using a structured questionnaire and tested for HIV, syphilis, gonorrhea, and Chlamydia.
The prevalence of syphilis, gonorrhea, and Chlamydia were 8.0% (4.6-12.2%), 9.5% (5.6-14.3%) and 3.9% (1.7-6.2%), respectively, and of any STIs was 19.7% (13.9- 26.2%). None of the participants was HIV-positive. The median number of clients during the previous week was 5. The proportion of consistent condom use with the clients during the previous week was 58.0% (50.4, 65.5%), use with regular non-paying partners and non-regular non-paying partners were much lower than that with clients. Multivariate analysis indicated that years of education and perception of HIV risk were protective factors. Awareness of HIV/AIDS, regular sex partners deciding about condom use when having sex, recruiting clients at hotels, hair/beauty salons, or streets instead of massage parlors, and higher charge for last sexual service were associated with STI infection.
Future intervention programs should not only address personal risk factors, but also empower FSWs to require condom use by both clients and non-paying partners.
PMCID: PMC2831231  PMID: 20104110
HIV/AIDS; STD; Sex workers; Behavior; China
17.  Growth of Human Colorectal Cancer SW1116 Cells Is Inhibited by Cytokine-Induced Killer Cells 
Previous reports have suggested that treatment with cytokine-induced killer (CIK) cells may benefit patients with various types of tumor. The aim of this study was to evaluate the antitumor effects of CIK cells against the colorectal cancer line SW1116 in vitro and in vivo. CIK cells were generated routinely from peripheral blood mononuclear cells of healthy human donors, and the number of CD3+CD56+ cells was expanded more than 1300-fold after 14-day culture. At an effector : target cell ratio of 50 : 1, the percentage lysis of SW1116 cells reached 68% in the presence of CIK cells, Experimental mice injected with SW1116 cells subcutaneously were divided randomly into four groups: untreated, 5-fluorouracil (5-FU)-treated, CIK-consecutive treated (injected once/day) and CIK-interval treated (injected once every 5 days). CIK cells were injected abdominally five times in total. Compared with the untreated group, xenograft growth was inhibited greatly by CIK treatment, to nearly the same extent as with 5-FU treatment. We demonstrated that the necrotic area in the tumor xenograft was markedly larger in the CIK-treated groups than in the other groups. These findings suggest that CIK-based immunotherapy may represent an effective choice for patients with colorectal cancer.
PMCID: PMC2997496  PMID: 21455282
18.  Keratin 18 attenuates estrogen receptor α-mediated signaling by sequestering LRP16 in cytoplasm 
BMC Cell Biology  2009;10:96.
Oncogenesis in breast cancer is often associated with excess estrogen receptor α(ERα) activation and overexpression of its coactivators. LRP16 is both an ERα target gene and an ERα coactivator, and plays a crucial role in ERα activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear.
Yeast two-hybrid screening, GST pulldown and coimmunoprecipitation (CoIP) identified the cytoplasmic intermediate filament protein keratin 18 (K18) as a novel LRP16-interacting protein. Fluorescence analysis revealed that GFP-tagged LRP16 was primarily localized in the nuclei of mock-transfected MCF-7 cells but was predominantly present in the cytoplasm of K18-transfected cells. Immunoblotting analysis demonstrated that the amount of cytoplasmic LRP16 was markedly increased in cells overexpressing K18 whereas nuclear levels were depressed. Conversely, knockdown of endogenous K18 expression in MCF-7 cells significantly decreased the cytoplasmic levels of LRP16 and increased levels in the nucleus. CoIP failed to detect any interaction between K18 and ERα, but ectopic expression of K18 in MCF-7 cells significantly blunted the association of LRP16 with ERα, attenuated ERα-activated reporter gene activity, and decreased estrogen-stimulated target gene expression by inhibiting ERα recruitment to DNA. Furthermore, BrdU incorporation assays revealed that K18 overexpression blunted the estrogen-stimulated increase of S-phase entry of MCF-7 cells. By contrast, knockdown of K18 in MCF-7 cells significantly increased ERα-mediated signaling and promoted cell cycle progression.
K18 can effectively associate with and sequester LRP16 in the cytoplasm, thus attenuating the final output of ERα-mediated signaling and estrogen-stimulated cell cycle progression of MCF-7 breast cancer cells. Loss of K18 increases the functional availability of LRP16 to ERα and promotes the proliferation of ERα-positive breast tumor cells. K18 plays an important functional role in regulating the ERα signaling pathway.
PMCID: PMC2804594  PMID: 20035625
19.  FHL2 interacts with and acts as a functional repressor of Id2 in human neuroblastoma cells 
Nucleic Acids Research  2009;37(12):3996-4009.
Inhibitor of differentiation 2 (Id2) is a natural inhibitor of the basic helix–loop–helix transcription factors. Although Id2 is well known to prevent differentiation and promote cell-cycle progression and tumorigenesis, the molecular events that regulate Id2 activity remain to be investigated. Here, we identified that Four-and-a-half LIM-only protein 2 (FHL2) is a novel functional repressor of Id2. Moreover, we demonstrated that FHL2 can directly interact with all members of the Id family (Id1–4) via an N-terminal loop–helix structure conserved in Id proteins. FHL2 antagonizes the inhibitory effect of Id proteins on basic helix–loop–helix protein E47-mediated transcription, which was abrogated by the deletion mutation of Ids that disrupted their interaction with FHL2. We also showed a competitive nature between FHL2 and E47 for binding Id2, whereby FHL2 prevents the formation of the Id2–E47 heterodimer, thus releasing E47 to DNA and restoring its transcriptional activity. FHL2 expression was remarkably up-regulated during retinoic acid-induced differentiation of neuroblastoma cells, during which the expression of Id2 was opposite to that. Ectopic FHL2 expression in neuroblastoma cells markedly reduces the transcriptional and cell-cycle promoting functions of Id2. Altogether, these results indicate that FHL2 is an important repressor of the oncogenic activity of Id2 in neuroblastoma cells.
PMCID: PMC2709579  PMID: 19417068
20.  Id2 promotes the invasive growth of MCF-7 and SKOV-3 cells by a novel mechanism independent of dimerization to basic helix-loop-helix factors 
BMC Cancer  2009;9:75.
Inhibitor of differentiation 2 (Id2) is a critical factor for cell proliferation and differentiation in normal vertebrate development. Most of the biological function of Id2 has been ascribed to its helix-loop-helix motif. Overexpression of Id2 is frequently observed in various human tumors, but its role for invasion potential in tumor cells is dispute. We aimed to reveal the role of Id2 in invasion potential in poorly invasive and estrogen receptor α (ERα)-positive MCF-7 and SKOV-3 cancer cells.
MCF-7 and SKOV-3 cells were stably transfected with the wild-type, degradation-resistant full-length or helix-loop-helix (HLH)-deleted Id2, respectively. Protein levels of Id2 and its mutants and E-cadherin were determined by western blot analysis and mRNA levels of Id2 and its mutants were determined by RT-PCR. The effects of Id2 and its mutants on cell proliferation were determined by [3H]-thymidine incorporation assay and the 3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye method. The in vitro invasion potential of cells was evaluated by Transwell assay. Cell motility was assessed by scratch wound assay. The promoter activity of E-cadherin was determined by cotransfection and luciferase assays.
Ectopic transfection of the wild-type Id2 markedly increased the protein and mRNA expression of Id2 in MCF-7 and SKOV-3 cells; the protein level but not mRNA level was further increased by transfection with the degradation-resistant Id2 form. The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells. Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2. E-cadherin protein expression and transactivation of the proximal E-cadherin promoter were markedly suppressed by the degradation-resistant full-length or HLH-deleted Id2 but not wild-type Id2. Ectopic expression of E-cadherin in MCF-7 and SKOV-3 cells only partially blunted the invasion potential induced by the degradation-resistant HLH-deleted Id2.
Overexpression of Id2 in ERα-positive epithelial tumor cells indeed increases the cells' invasive potential through a novel mechanism independent of dimerization to basic helix-loop-helix factors. E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.
PMCID: PMC2654660  PMID: 19257909
21.  Intradiscal methylene blue injection for the treatment of chronic discogenic low back pain 
European Spine Journal  2006;16(1):33-38.
This article was a preliminary report of prospective clinical trial of a group of patients with chronic discogenic low back pain who met the criteria for lumbar interbody fusion surgery but were treated instead with an intradiscal injection of methylene blue (MB) for the pain relief. Twenty-four patients with chronic discogenic low back pain underwent diagnostic discography with intradiscal injection of MB. The principal criteria to judge the effectiveness included alleviation of pain, assessed by visual analog scale (VAS), and improvement in disability, as assessed with the Oswestry Disability Index (ODI) for functional recovery. The mean follow-up period was 18.2 months (range 12–23 months). Of the 24 patients, 21 (87%) reported a disappearance or marked alleviation of low back pain, and experienced a definite improvement in physical function. A statistically significant and clinically meaningful improvement in the changes in the ODI and the VAS scores were obtained in the patients with chronic discogenic low back pain (P=0.0001) after the treatment. The study suggests that the injection of MB into the painful disc may be a very effective alternative for the surgical treatment of chronic discogenic low back pain.
PMCID: PMC2198898  PMID: 16496191
Discogenic low back pain; Discography; Methylene blue; Injection

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